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WO1994022465A1 - Procede de reduction de la viscosite de mucosites pathologiques presentes dans les voies respiratoires - Google Patents

Procede de reduction de la viscosite de mucosites pathologiques presentes dans les voies respiratoires Download PDF

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Publication number
WO1994022465A1
WO1994022465A1 PCT/US1994/003667 US9403667W WO9422465A1 WO 1994022465 A1 WO1994022465 A1 WO 1994022465A1 US 9403667 W US9403667 W US 9403667W WO 9422465 A1 WO9422465 A1 WO 9422465A1
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Prior art keywords
actin
gelsolin
binding
mucus
dnase
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PCT/US1994/003667
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English (en)
Inventor
Thomas P. Stossel
Stuart E. Lind
Paul A. Janmey
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Brigham And Women's Hospital
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Publication date
Priority to JP6522448A priority Critical patent/JPH08510998A/ja
Priority to GB9520020A priority patent/GB2293102B/en
Priority to CA002159205A priority patent/CA2159205A1/fr
Priority to AU66250/94A priority patent/AU695033B2/en
Priority to BR9405856A priority patent/BR9405856A/pt
Priority to DE69419907T priority patent/DE69419907T2/de
Application filed by Brigham And Women's Hospital filed Critical Brigham And Women's Hospital
Priority to KR1019950704203A priority patent/KR100308679B1/ko
Priority to DK94914023T priority patent/DK0692970T3/da
Priority to EP94914023A priority patent/EP0692970B1/fr
Publication of WO1994022465A1 publication Critical patent/WO1994022465A1/fr
Priority to NO953862A priority patent/NO953862D0/no
Priority to GR990402778T priority patent/GR3031753T3/el

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/12—Mucolytics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to the treatment of respiratory disorders in which it is desired to disaggregate, depolymerize, or solubilize actin filaments or to promote the degradation of DNA in respiratory tract mucus.
  • the invention particularly relates to solubilization and increased clearance of mucus obstructing the respiratory tract in airway diseases.
  • the invention relates to a method for reducing the viscosity of mucus by instillation of actin-binding proteins into the respiratory tract.
  • the invention also relates to a method for facilitating the action of DNase I in respiratory tract treatments by blocking actin binding to DNase I or reducing the levels of actin in respiratory tract mucus.
  • the method involves the administration of efficacious amounts of actin-binding compounds, or fragments or functional derivatives thereof, such that the compounds or fragments or derivatives are active in the respiratory tract of a patient in need of such treatment.
  • Airway obstruction caused by inflammatory exudation is a major cause of morbidity and mortality.
  • Mucus plugging and stasis is a feature of chronic bronchitis, asthmatic bronchitis, bacterial bronchopneumonia and, especially, cystic fibrosis, and is associated with destruction of lung substance. Mucus also contributes to the morbidity of acute and chronic sinusitis and even the common cold.
  • Mucoid obstruction of the respiratory system is multifactorial.
  • One factor is increased mucus synthesis and release caused by inflammatory mediators elicited by infection or irritation.
  • cystic fibrosis the viscosity of mucus is increased, presumably because of abnormal epithelial ion transport which affects the hydration or charge of the ionic polymers composing the mucus substance.
  • the thick mucus of cystic fibrosis prevents bacterial clearance by ciliary transport and favors the growth of bacteria, especially Pseudomonas aeruginosa .
  • These bacteria or other irritants such as tobacco smoke, generate chemoattractants which recruit leukocytes into the airway. As the leukocytes engage bacteria or irritants, they degenerate and their components contribute debris that affect the viscoelasticity of the airway contents.
  • Purulent mucus contains about 10-13 mg/ml of DNA, an ionic polymer widely believed to affect the rheologic properties of airway fluids.
  • Bovine pancreatic DNase I an enzyme that degrades DNA, was tested as a mucolytic agent many years ago. However, it did not enter clinical practice due to side effects induced by antigenicity and/or contaminating proteases. Accordingly, cDNA for human DNase I was cloned and expressed. Recombinant human DNase I was tested as a therapeutic agent.
  • Actin Extracellular Actin Actin is the most abundant protein in nucleated animal cells and constitutes 10-20% of the protein of many nucleated cells and 30% of the protein of muscle cells. Actin molecules each bind an ATP or ADP molecule and self-assemble into long filaments during which the ATP is hydrolyzed into ADP.
  • actin the double-helical, rodlike, filament form of actin which is assembled from G-actin monomers
  • F-actin the double-helical, rodlike, filament form of actin which is assembled from G-actin monomers
  • the ionic conditions of extracellular fluids favor actin polymerization, so that virtually all the actin released from dying cells would be expected to polymerize into filaments if sufficiently concentrated (greater than a few micrograms per milliliter) (Lind, S.E. et al . , Am . Rev. Respir. Dis . 138:429-434 (1988)) .
  • Actin polymerizes into long, rod-like filaments which, are relatively resistant to degradation by proteolytic enzymes. In purified solutions, in the absence of filament-shortening proteins, actin filaments can easily attain lengths of several microns.
  • actin from dying cells provides sufficient actin to have a significant effect on the microenvironment, either by increasing the viscosity of extracellular fluids, such as mucin, and/or by entrapping cells or by other, as yet unidentified toxic effects. Infusion of extracellular free actin is toxic to animal tissues (Harper, K.D. et al., Clin . Res .
  • actin released from injured cells were to be bound to an actin-binding protein, such as those discussed below, this actin would remain monomeric or oligomeric.
  • Plasma gelsolin (sometimes called brevin) and DBP (also called Gc globulin) are two high affinity actin-binding proteins that exist in plasma. High affinity actin-binding proteins bind actin with a K d of less than IO" 8 . Both gelsolin and DBP bind to actin in blood serum and have actin depolymerizing activity. DBP preferentially binds monomeric actin while gelsolin preferentially binds actin filaments. Gelsolin is a multifunctional actin-binding protein obtained from mammalian cytoplasm and extracellular fluids. Plasma gelsolin differs from cellular gelsolin by an additional 25 amino acids at the amino terminus of the molecule. Both gelsolins are the products of a single gene. Plasma gelsolin has three actin-binding sites and binds with high affinity to either G-actin or F-actin.
  • Plasma gelsolin binds a second actin molecule with a higher affinity than it binds a first actin molecule. Thus, it preferentially forms 2:1 complexes over 1:1 complexes and binds filaments in preference to monomers.
  • plasma gelsolin severs the filament in a nonproteolytic manner and remains bound to one end of the newly formed filament. If free gelsolin molecules are present, they will sever the actin filament successively until only 2:1 actin- gelsolin complexes are present, thereby rapidly depolymerizing the filament.
  • Free and complexed (to actin) gelsolin molecules differ in their functional properties. Although free gelsolin can sever actin filaments, actin-gelsolin complexes cannot.
  • Gelsolin's primary function in the plasma and other extracellular fluids is to sever actin filaments. If gelsolin is present in excess of actin, only gelsolin-actin complexes are formed; if actin is in excess, there are free actin oligomers and gelsolin- actin complexes. The actin severing occurs by way of a nonproteolytic cleavage of the noncovalent bond between adjacent actin molecules.
  • Gelsolin's severing activity is activated by micromolar Ca ++ and by pH above 6, and has been shown to be inhibited by phosphatidyl inositol 4-5-bisphosphate (PIP 2 ) and phosphatidyl inositol-4- monophosphate (PIP) . Since extracellular Ca ++ concentrations are at millimolar levels and extracellular fluids do not normally contain PIP or PIP 2 in a form that inhibits gelsolin, plasma gelsolin is constitutively active in extracellular fluids.
  • Plasma gelsolin has been detected in many body fluids, suggesting that gelsolin may also be a normal component of airway fluid, at least in states of inflammation.
  • DBP has also been cloned (Cooke, N.E. et al . , J. Clin . Invest . 76:2420-2424 (1985); Yang, F. et al . , Proc . Natl . Acad. Sci . USA 82:7994-7998 (1985)). DBP has a single actin-binding site and binds constitutively to monomeric but not F-actin. Actin and DNase I
  • the catalytic activity of DNase I is inhibited by actin, to which the enzyme binds tightly (Lindberg, U. , Biochem . Biophys . Acta 82:237-248 (1964) ; Lazarides and Lindberg, Proc . Natl . Acad . Sci . USA 71:4742-4746 (1974)) .
  • Actin comprises about 10% of the protein mass of leukocytes. It follows, therefore, that abundant actin, as well as DNA polymers, might exist in purulent airway contents. If actin does exist in these contents, the efficacy of DNase I in DNA degradation would predictably be reduced by binding to actin present in the very secretions for which DNase I was intended.
  • Actin filaments alone produce networks of high elasticity, and when interpenetrated within mucus and/or DNA polymers, predictably would contribute to very rigid gels as they do with fibrin (Janmey, P.A., Blood 80:928-936 (1992)) .
  • Introduction of actin- binding molecules that disaggregate actin filaments, especially by severing actin filaments is therefore an attractive approach to reducing the consistency of airway mucus, and especially purulent airway mucus.
  • actin-binding molecules should also enhance the efficacy of DNase I in degrading DNA by solubilizing actin and facilitating its removal from DNA-containing airway mucus in which DNase I is intended to be effective.
  • the present invention is based upon the Applicants' consideration that, since leukocytes are a significant component of purulent mucus and actin comprises about 10% of the protein mass of leukocytes, abundant actin might exist in purulent mucoid airway contents.
  • the invention is also based on the consideration that the efficacy of DNase I in degrading the DNA polymers in purulent mucus would predictably be reduced by binding to actin present in the mucus. Therefore, the effect of both endogenous DNase I and DNase I added for therapeutic purposes would be reduced by binding to actin present in the very secretions for which the enzymes were intended.
  • the present invention is based upon the Applicant's consideration that the administration of actin-binding compounds, or fragments or functional derivatives thereof, to patients with pathological mucoid airway contents will provide treatment of and protection against obstruction of the respiratory tract. It is therefore an object of the invention to provide a method for depolymerizing actin filaments, preventing actin polymerization, and for decreasing the levels of actin in the respiratory tract of individuals with airway disease.
  • the invention provides a method to reduce the viscosity or consistency of pathological airway contents by instillation into the respiratory tract of actin-binding compounds, and especially actin- binding proteins which: 1) disaggregate (depolymerize, fragment and dissociate) or prevent aggregation of actin filaments released from disrupted leukocytes and other cells that solidify airway contents, and especially mucoid airway contents, and/or 2) prevent actin from inhibiting the action of the enzyme DNase I which degrades DNA polymers that increase the viscoelasticity of mucus.
  • the DNase I may be endogenous or supplied for therapeutic purposes by aerosolization or any other appropriate and effective means.
  • a further object of the invention is reducing the level of actin binding to DNase I in the respiratory tract by the administration of actin-binding compounds, and especially actin- binding proteins, to the respiratory tract.
  • a further object of the invention is to enhance DNA degradation in pathological mucoid airway contents by the administration of actin-binding compounds, and especially actin-binding proteins.
  • FIG. 1 Dynamic shear moduli of cystic fibrosis sputum .
  • Sputum samples with or without gelsolin or gelsolin fragment were placed between the parallel circular plates of a torsion pendulum (Ferry, J.D. Viscoelastic Properties of Polymers, 3rd Ed., John Wiley & Sons, New York (1980)) and a momentary displacement was applied to the arm attached to the upper sample plate.
  • the dynamic shear modulus, G ' was calculated from the frequency and damping of the resulting free oscillations.
  • G ' of actin polymer- containing solutions depends strongly on the average actin filament length (Janmey et al . , Biochemistry 27:8218-8226 (1988)) .
  • IA Cystic fibrosis sputum alone
  • IB Cystic fibrosis sputum plus gelsolin
  • 1C Cystic fibrosis sputum plus gelsolin amino acid fragment 1-260.
  • Figure 2 Shear creep and recovery of cystic fibrosis sputum .
  • Sputum samples with or without gelsolin or gelsolin fragment placed between plates of the torsion pendulum were subjected to a constant shear stress.
  • the subsequent deformation is quantified as the shear compliance, the ratio of shear strain to the imposed stress.
  • Previous studies have shown that the compliance of F-actin increases sharply as the average filament length decreases (Janmey et al . , Biochemistry 27:8218-8226 (1988)).
  • the abrupt decreases in strain represent release of the applied stress.
  • Figure 3 Dose response curve for gelsolin and DNase I. The change in viscosity of sputum samples with gelsolin or DNase I was measured in a torsion pendulum after 60 minutes of incubation. The change in viscosity with increasing concentration is shown as a percentage of the initial viscosity.
  • Figure 4 Effect of gelsolin combined wi th
  • Figure 5 Effect of a combination of actin- binding compounds on the viscosi ty of sputum .
  • the change in viscosity of sputum samples after the addition of gelsolin combined with Gc globulin (vitamin D binding protein) was measured in a torsion pendulum over a period of 60 minutes after the addition of the compounds. The results are shown as a percentage of the initial viscosity over time.
  • an object of the invention is to reduce the viscosity or consistency of pathological airway contents by causing the disaggregation or depolymerization of actin filaments, the inhibition of actin polymerization, reduction of actin levels, and the degradation of DNA polymers.
  • an actin-binding compound or compounds are introduced into the respiratory tract of a patient whose airway contents contain actin filaments and DNA polymers that contribute to respiratory distress.
  • the actin-binding compound may disaggregate actin, prevent or reduce the polymerization of free monomeric actin, and/or prevent the binding of actin filaments or monomers to DNase I.
  • a general embodiment of the invention is a method for the solubilization or reduction in consistency or viscosity ⁇ f pathological respiratory contents in the respiratory tract of individuals experiencing respiratory distress as a result of the presence of such contents.
  • airway mucus may contain actin filaments and DNA polymers which increase the consistency or viscosity of this mucus and thus prevent normal airflow and clearance. Accordingly, it is an object of the present invention to reduce the viscosity or consistency of such mucus by the administration, into the respiratory tract containing such mucus, of an actin-binding compound or compounds.
  • the compound may act by disaggregating (depolymerizing) actin filaments into actin monomers, by binding to actin so as to prevent the binding of actin to DNase I, or both.
  • the compound may also act by preventing the polymerization of actin monomers into filaments. By promoting mucus clearance, the compound ultimately can reduce the levels of actin in the airway contents.
  • a method for reducing the consistency or viscosity of airway mucus or solubilizing said mucus, by the introduction of an actin-binding compound or compounds into the respiratory tract of an individual whose respiratory tract contains said mucus.
  • the present invention is further based upon the consideration that purulent mucus in the respiratory tract contains significant leukocyte contamination such that said mucus contains DNA polymers increasing the consistency or viscosity of said mucus and may contain amounts of actin filaments such that the mucus contributes to airway obstruction, reduced expectoration, and other forms of respiratory distress. Accordingly, it is an object of the invention to reduce the consistency or viscosity of purulent mucus by causing the disaggregation (depolymerization) of actin filaments that may be present in said mucus and by enhancing the degradation of DNA polymers present in said mucus.
  • the consistency or viscosity of purulent mucus is reduced by introducing into the respiratory tract of an individual with said mucus, an actin-binding compound or compounds.
  • the actin-binding compounds may produce their effect by depolymerizing actin into actin monomers, binding to actin filaments and monomers so as to prevent their binding to DNase I, or both.
  • the compound may also act by preventing the polymerization of actin monomers into filaments. By promoting mucus clearance, the compound ultimately can reduce the levels of actin in the airway contents.
  • DNase I is present, it can be either endogenous or added exogenously for therapeutic purposes. When it is added for therapeutic purposes, it may be co- administered with the actin-binding compound or compounds or separately.
  • fragments or functional derivatives of the actin- binding compounds are administered to patients with pathological airway contents, mucus, or purulent mucus in their respiratory tracts to protect against obstruction of the respiratory tract or reduction of expectoration.
  • the methods are directed to reducing the consistency of the specific airway contents by introduction into the respiratory tract of a patient in need of such treatment, actin-binding compounds which may do one or more of the following: (1) disaggregate, depolymerize, fragment, and dissociate actin filaments (or prevent aggregation or polymerization of actin monomers and oligomers) released from disrupted leukocytes and other cells that solidify airway contents; (2) prevent actin from inhibiting the action of DNase I which degrades DNA polymers by blocking actin binding to DNase I; (3) ultimately reduce the level of actin in airway contents by promoting clearance of the contents.
  • actin present in respiratory contents for the purpose of the invention, is extracellular actin in said contents in free monomer or filament form.
  • the methods are practiced with the protein gelsolin or an active fragment or functional derivative thereof.
  • Gelsolin may be introduced into the respiratory tract by routine methods such as those used to administer DNase I to patients with respiratory distress. The treatment is not, however, limited to gelsolin alone. Gelsolin may be co-administered with other actin-binding compounds (preferably vitamin D binding protein) or with DNase I.
  • an actin-binding compound or compounds may also be introduced into the airway by the intravenous route.
  • an actin-binding compound or compounds preferably gelsolin
  • the embodiment is based on the consideration that drug delivery by inhalation may be inherently limited in congested airways because of obstruction. Effective solubilization could be expected to enhance drug delivery by inhalation.
  • an assault from the blood side may deliver the drug into alveoli as well as bronchioles and bronchi, such delivery being heightened by chronic inflammation and concomitant increased blood-tissue permeability.
  • plasma proteins can enter the airways as evidenced by ⁇ .-1-antitrypsin therapy of hereditary emphysema. Presumably the efficiency of blood to tissue delivery of plasma proteins is a direct function of blood levels of those proteins. Accordingly, plasma gelsolin levels can be measured to determine if airway binding to actin has had a depleting effect. It was shown (Am . J. Path . 130:261-267 (1988) and Am. .Rev. Resp . Dis .
  • the ultimate object of the embodiment is to enhance DNA degradation in pathological airway contents, and particularly in mucus and purulent mucus.
  • the diseases that are amenable to treatment by the methods of the present invention include, but are not limited to cystic fibrosis, chronic bronchitis, mucopurulent or purulent exacerbation of simple mucoid bronchitis, bronchorrhea, bronchopneumonia, widespread bronchiolitis, purulent pneumonia, pneumonic-alveolar- consolidation, asthma, with or without asthmatic bronchitis with mucus plugging, acute and/or chronic purulent sinusitis, empyema, bronchiectasis, bronchocoele, adult respiratory distress syndrome (ARDS) , post-transplantation obliterative bronchiolitis, and allergenic bronchiolitis (fibrosing alveolitus) .
  • cystic fibrosis chronic bronchitis
  • mucopurulent or purulent exacerbation of simple mucoid bronchitis bronchorrhea
  • bronchopneumonia widespread bronchiolitis
  • the term "respiratory tract” or "airway” would be understood by the skilled artisan to refer to the tubular and cavernous organs and structures by means of which pulmonary ventilation and gas exchange between ambient air and blood are brought about. This would be understood to include, inter alia , the nasal cavity and conchae, the pharyngeal opening of the auditory tube, the pharynx, the larynx, the trachea, the bronchi, and the lungs.
  • the invention is directed to treatment of airway contents, mucus, purulent mucus, etc. in any of the parts of the tract, particularly sub-tracheal.
  • airway contents or "respiratory tract contents” comprise a mixture of substances normally produced by the cells lining the airway (for example, through secretion) or remnants of the cells themselves.
  • pathological (mucoid) airway contents or “pathological (mucoid) contents of the respiratory tract” to refer to those contents that contain components leading to structural and functional changes in the airway contents (such as excess actin) which cause abnormal respiratory tract function and/or disease. These include, but are not limited to, mucus, purulent mucus, thick mucus, such as in cystic fibrosis, bacteria, and the like.
  • the components may also be biological components of mucus that alter the properties of mucus and cause disease or distress.
  • the components themselves may be caused by an underlying disease state that contributes to an increase in the amount of normal airway contents or a change in the type of components from the components normally found in airway contents. For example, there may be an imbalance or disproportionate amount of certain components normally found in airway contents (e.g., excess actin) .
  • respiratory distress is meant the consequences of pathological airway contents which include, but are not limited to, reduced airflow, obstruction of the airway, reduced respiratory clearance by cilia, reduced respiratory clearance by muscular mechanisms such as coughing, clearing the throat, and sneezing, respiratory irritation from prolonged contact with the contents, the retention of leukocyte-derived lytic enzymes, inflammatory mediators, fungi, mycoplasma, bacteria, or other microorganisms.
  • mucus the ordinary skilled artisan would understand the free slime of the mucus membranes, composed of secretion of the glands, along with various inorganic salts, desquamated cells, and leukocytes.
  • mucus that is rendered highly viscous, relatively solidified by DNA polymers derived from white blood cells and/or desquamated and/or broken endogenous airway cells.
  • viscosity would be understood by the skilled artisan to denote a physical property of a substance that is dependent on the friction of its component molecules as they slide by one another.
  • a highly viscous solution would be characterized by a high degree of friction between component molecules, whereas a reduced viscosity would be characterized by a decrease in the degree of friction between component molecules as they slide by each other.
  • exogenous DNase is intended DNase which is added by clinical means to a patient in need of such DNase by virtue of respiratory distress caused by DNA polymers in the patient's respiratory tract.
  • endogenous DNase is intended DNase which is found normally in the respiratory tract of a patient, and particularly in the mucus of said patient's respiratory tract.
  • inhibiting this DNase from reducing the amount of DNA polymer to allow decreased viscosity, enhanced respiratory airflow, expectoration, and relief from other forms of respiratory distress caused by the airway contents.
  • actin-binding compound is meant to include any compound, and especially any protein (or peptide) , which is capable of binding actin so as to modify any of actin's many functions, including suppressing the ability of actin monomers to polymerize into filaments, to fragment (disaggregate, depolymerize) pre-existing filaments, and to bind to DNase I.
  • actin-binding compounds of the invention are substantially free of natural contaminants which . associate with such compound either in vivo (in a prokaryotic or eukaryotic) host, or in vi tro (as a result of a chemical synthesis) .
  • Such compounds include, but are not limited to extracellular actin- binding proteins such as gelsolin and DBP, and intracellular actin-binding proteins such as those most abundant in cells (for example, myosins, tropomyosins, profilin and cofilin) and those most abundant in non- muscle cells.
  • Actin-binding compounds within the scope of the methods of the invention also include but are not limited to a) actin-binding compounds that predominantly sequester actin monomers, that is, bind monomers in a complex which is resistant to polymerization (for example, DBP, profilin, 3 4 -thymosin
  • actin-binding compounds which sequester monomers and possess filament severing activity (for example, gelsolin, villin, fragmin and severin; c) actin-binding compounds that predominantly block the ends of actin filaments and prevent the exchange of monomers with that end (for example, tropomodulin, capZ, cap 100, ASP-56) ; and d) actin- binding nonproteinaceous molecules that have such effects on actin (for example, cytochalasin or biologically-active derivatives thereof, that block the ends of actin filaments and which are modified to remain extracellular) .
  • actin-binding compounds which sequester monomers and possess filament severing activity (for example, gelsolin, villin, fragmin and severin; c) actin-binding compounds that predominantly block the ends of actin filaments and prevent the exchange of monomers with that end (for example, tropomodulin, capZ, cap 100, ASP-56) ; and d) actin- binding nonproteinace
  • Fragments of actin-binding proteins with retained actin filaments severing and/or monomer-sequestering activity are also within the scope of the present invention. If desired, such compounds may be administered in the form of a pharmaceutically acceptable salt to the animal.
  • animal is meant to include all animals in which free actin or actin filaments in the sputum would be detrimental to the physiology of the animal. Foremost among such animals are humans; however, the invention is not intended to be so limiting, it being within the contemplation of the present invention to treat any and all animals which may experience the beneficial effect of the invention.
  • An "efficacious amount" of an actin-binding compound is one which is sufficient to reduce or eliminate the toxic effects of actin in the respiratory tract contents of an animal .
  • an efficacious amount of an actin-binding compound is also that amount which may reduce bacterial content, the incidence of clinical infection, reduce the content of inflammatory mediators, or the content of degradative and cytotoxic enzymes in respiratory distress syndromes causing or caused by these liquids or gels.
  • the amount of the actin-binding molecule to be given, and the duration of therapy may be determined by monitoring the frequency of exacerbation of symptoms or the deterioration of symptoms, the sputum volume and consistency, the pulmonary functions such as FEV, blood p0 2 , pH, pC0 2 , total lung capacity, residual volume tests of air flow, infection rate, etc.
  • the skilled clinician would be familiar with routine ⁇ monitoring techniques for respiratory treatment.
  • the amount of aerosolized actin-binding protein may be determined according to those procedures used to determine the doses of DNase inhalation effective for treatment of patients with DNase I. For example, see Aitken, M.L., et al . , J. Am. Med. Assoc .
  • the dose delivered in vivo is related to the effective amount determined in vi tro .
  • sputum is successfully solubilized in vi tro by the amounts exemplified herein (i.e., 2-10 ⁇ g/ l)
  • a thousand-fold increase over the in vi tro dose would be administered (i.e., 2-10 mg/ml) .
  • the amounts and regimens for the administration of actin-binding compounds can be determined readily by those of ordinary skill in the art of treating respiratory disorders.
  • the dosage of actin-binding compound treatment will vary depending upon considerations such as the type of the actin-binding compound employed, the age of the patient, the health of the patient, the severity of the disease, the kind of concurrent treatment, the extent of tissue damage, the gender of the patient, the duration of the symptoms, counterindications (if any) , and other variables understood as needing adjustment by the individual physician.
  • the correct effective dose could routinely be determined by monitoring the airway contents by aspiration or as sputum.
  • a material is said to be “substantially free of natural contaminants if it has been substantially purified from materials with which it is normally and naturally found before such purification.
  • natural contaminants with which actin-binding compounds might be associated are: non-actin-binding peptides, carbohydrates, glycosylated peptides, lipids, membranes, etc.
  • a material is said to be substantially free of natural contaminants if those contaminants normally and naturally found with the substance in vivo or in vi tro are substantially absent from a sample of the material.
  • substantially absent is meant that such contaminants are either completely absent or are present at such low concentrations that their presence (1) does not interfere with the desired therapeutic effect of the active agent (herein the actin-binding compound) in the preparation when such preparation is administered to an animal and (2) does not harm the animal as the result of the administration of such preparation.
  • active agent herein the actin-binding compound
  • administration is meant to include introduction of actin-binding compounds into the respiratory tract of an animal by any appropriate means known to the medical art, including, but not limited to inhalation of an aerosolized compound, instillation by bronchoscopy, and intravenous infusion.
  • actin-binding compounds of the present invention may be co-administered with DNase or one actin-binding compound may be co-administered with one or more distinct actin-binding compounds.
  • co-administer is intended that each of at least two compounds be administered during the time frame wherein the respective periods of biological activity overlap.
  • the term includes sequential as well as coextensive administration of the compounds of the present invention with each other or with DNase I.
  • pharmaceutically acceptable vehicle is intended to include solvents, carriers, diluents, and the like, which are utilized as additives to preparations of the actin-binding compounds of the invention so as to provide a carrier or adjuvant for the administration of such compounds.
  • treatment or “treating” is intended to include the administration of actin-binding compounds to a patient for purposes which may include prophylaxis, amelioration, prevention or cure.
  • fragment is meant to include any portion of a molecule which provides a segment of an actin-binding compound which is capable of binding actin monomers and/or severing filaments; the term is meant to include actin-binding fragments which are made from any source, such as, for example, from naturally- occurring peptide sequences, synthetic or chemically- synthesized peptide sequences, and genetically engineered peptide sequences. Further, if such fragment is a peptide, a fragment of a peptide of such actin-binding protein is meant to include to any variant of the actin-binding protein.
  • a “functional derivative” of an actin-binding compound is a derivative which possesses a biological activity that is substantially similar to the biological activity of the actin-binding compound.
  • substantially similar is meant activity which is quantitatively different but qualitatively the same.
  • a functional derivative of an actin- binding protein of the invention would contain the same amino acid backbone as an actin-binding protein but also contains other modifications such as post- translational modifications such as, for example, bound phospholipids, or covalently linked carbohydrate, depending on the necessity of such modifications for the performance of the diagnostic assay or therapeutic treatment.
  • the term is also meant to include a chemical derivative of an actin-binding compound.
  • Such derivatives may improve the compound's solubility, absorption, biological half life, etc.
  • the derivatives may also decrease the toxicity of the molecule, or eliminate or attenuate any undesirable side effect of the molecule, etc.
  • Derivatives and specifically, chemical moieties capable of mediating such effects are disclosed in Remington ' s Pharmaceutical Sciences (1980) . Procedures for coupling such moieties to a molecule are well known in the art.
  • the term "functional derivative” is intended to include chemical derivatives, variants, analogs, or pharmaceutically acceptable salts of an actin-binding compound.
  • a “variant” is meant to refer to a compound substantially similar in structure and biological activity to either the native compound, or to a fragment thereof.
  • an "analog" of an actin-binding compound of the invention is meant to refer to a compounds substan ⁇ tially similar in function to either the native actin- binding compound or to a fragment thereof.
  • an analog of an actin-binding protein is a protein which does not have the same amino acid sequence as an actin-binding protein but which is sufficiently homologous to an actin-binding protein so as to retain the biological activity of such actin- binding protein.
  • salts of the actin-binding compounds of the invention can be formed from pharmaceutically acceptable acids or bases, such as, for example, acids such as sulfuric, hydrochloric, nitric, phosphoric, etc., or bases such as alkali or alkaline earth metal hydroxides, ammonium hydroxides, alkyl ammonium hydroxides, etc.
  • the "biological activity" of the compounds of the invention is their ability to bind actin and modify it into a form which is less toxic to an animal than • unmodified actin. Such modification may be the result of the binding of the compounds per ⁇ e or the result of a chemical or enzymatic reaction which results from such binding.
  • actin-binding molecules that are the subject of the methods of the invention are purified native and recombinant actin-binding proteins, and other non-proteinaceous actin-binding molecules, and biologically-active fragments thereof, which are characterized by the presence of unique actin-binding domains which possess the biological activity of being able to sequester actin in a monomeric form or rapidly to desegregate or depolymerize actin filaments or to cover sites on free actin that are toxic to host cells.
  • Individual actin-binding domains possessing this biological activity may also be produced by synthetic, enzymatic, proteolytic, chemical or recombinant DNA methods.
  • gelsolin or actin- binding fragments thereof, or DBP, a combination of gelsolin or actin-binding fragments thereof and DBP are provided to a patient in need of treatment.
  • Other preferred actin-binding compounds for use in this invention include ⁇ 4 thymosin and ASP-56.
  • gelsolin or actin- binding fragments thereof, or, a combination of gelsolin or actin-binding fragments thereof and vitamin D binding protein are co-administered with DNase I to a patient in need of treatment.
  • Preparations of the actin-binding proteins of the invention for parenteral administration include sterile aqueous or non-aqueous solvents, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
  • Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose and the like.
  • actin-binding proteins of the invention may also be administered by means of pumps, or in sustained-release form, especially, when the primary injury is prolonged or delayed rather an acute.
  • Administration in a sustained-release form is more convenient for the patient when repeated injections for prolonged periods of time are indicated.
  • actin-binding proteins of the invention can be employed in dosage forms such as tablets, capsules, powder packets, or liquid solutions for oral administration if the biological activity of the protein is not destroyed by the digestive process and if the characteristics of the compound allow it to be absorbed across the intestinal tissue.
  • compositions of the present invention are manufactured by means known to those of skill in the art, for example, by means of conventional mixing, granulating, dragee-making, dissolving, lyophilizing or similar processes.
  • the compositions of the present invention in and of themselves, find utility in the control of actin- induced physiological damage, be it chronic or acute.
  • the compositions of the invention direct the body's own mechanisms for dealing with excess actin in the bloodstream or extracellular tissues to its maximum potential.
  • the compositions of the present invention In intravenous dosage form, the compositions of the present invention have a sufficiently rapid onset of action to be useful in the acute management of potential tissue damage.
  • Actin-binding proteins which are substantially free of natural contaminants can be isolated and purified from their natural or recombinant sources in accordance with conventional conditions and techniques in the art previously used to isolate such proteins, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • actin-binding domain(s) of an actin-binding compound can be identified using techniques known in the art, without undue experimentation, and such domains are preferred in the methods of the invention.
  • derivatives of the native actin-binding proteins, or, derivatives of recombinantly produced actin-binding proteins can be made by proteolytic cleavage of the full-length actin- binding protein with common proteases, such as, for example, trypsin, chymotrypsin, and subtilisin.
  • Affinity chromatography with actin-derivatized resins may be used to assay such fragments for their actin- binding ability.
  • such compounds or fragments thereof which possess actin-severing activity can also be identified using techniques known in the art, for example, by following the rate of depolymerization of • pyrene-labeled F-actin.
  • fragments may be identified by their homology to other known actin-binding or actin- severing domains wherein it may be predicted that function will follow homology. For example, it is known that severin, gelsolin and villin, and especially amino acid residues 40-351 in severin and amino acid residues 63-383 in gelsolin, show extensive homology in the domain responsible for F-actin severing activity.
  • the N-terminal half of gelsolin for example, an N-terminal tryptic fragment known as CT45, is capable of severing F-actin and contains two actin binding sites. Efficacious amounts of CT45 which are substantially free of natural contaminants can be administered to a patient. One of these sites resides in a chymotryptic fragment, CT15N (human gelsolin residues 24-150) , which binds the ends of actin monomers and filaments with high affinity; the other site is contained in the adjacent fragment CT28N (residues 151-406) , which binds to the side of F-actin in a polyphosphoinositide-regulated manner. Neither of the fragments sever actin filaments by themselves. The smallest gelsolin polypeptide which is capable of severing F-actin encompasses residues 25-165 of plasma gelsolin.
  • actin-binding proteins are highly conserved among species and can be easily isolated in large quantities from nonhuman (bovine, porcine) plasma and/or muscle tissues and fragments of these proteins can be chemically or enzymatically prepared by techniques well-known in the art.
  • actin-binding compounds can be administered to a patient in need of the therapeutic methods of the invention without provoking a severe immune response.
  • Rhodamine phalloidin which binds to polymeric actin, was added to cystic fibrosis sputum specimens which were then observed in the phase contrast and fluorescence microscopes.
  • the samples contained cellular debris, the presumed source of DNA and actin, and were brightly fluorescent.
  • Untreated and gelsolin-fragment-treated sputum plugs were diluted in 2.5 ml of 0.15 M NaCl solution and centrifuged. The absorbance at 280 nm was determined as an indication of how much protein was released from the sputum gels. The results are shown in Table 2 and show that gelsolin and the 260 kDa fragment released protein from the sputum plugs.
  • Viscosity was measured in a torsion pendulum and values were computed from the rate of creep of the sample at a stress of 125 mPa, as described in P.A. Janmey, J. Biochem. Biophvs. Methods. 22:41 (1991). Uniform plugs of .25 ml were cut from coherent masses of the sputum samples and the plugs were layered onto round cover-slips. Ten microliters of Buffer A without additives or Buffer A with 50, 75 150, 250 or 500nM purified, human gelsolin or with 100, 250, 500, 600 or 750 nM, bovine pancreatic DNase I (Worthington Biochemical, Freehold, New Jersey) were added to the sputum samples. After a 60-minute incubation, the viscosity of the sputum sample from each patient was measured in duplicate at each concentration of additive.
  • gelsolin caused a marked decrease in viscosity even at the lowest concentration.
  • DNase I had no effect on the viscosity of the sputum at concentrations of up to 500nM.
  • the A 280 readings were as follows: 2 ⁇ g/ml: 0.25; 4 ⁇ g/ml: 1.30; 7 ⁇ g/ml: 1.86; 21 ⁇ g/ml: 180.
  • the A 280 after 4 ⁇ g/ml DNase I was approximately 0.5.
  • the A 280 after 4 ⁇ g/ml DNase I plus 2 ⁇ g/ml gelsolin was approximately 1.3. Gelsolin produced more dissolution expected at a low dose in the presence of a basally ineffective DNase I concentration.
  • Viscosity was measured in a torsion pendulum and values were computed from the rate of creep of the sample at a stress of 125 mPa, as described in P.A. Janmey, J. Biochem. Biophvs. Methods. 22:41 (1991). Uniform plugs of .25 ml were cut from coherent masses of the sputum samples and the plugs were layered onto round cover-slips. Ten microliters of Buffer A without additives, with 250 nM purified human gelsolin, 250 nM bovine pancreatic DNase I (Worthington Biochemical, Freehold, New Jersey) or with a 250nM combination of gelsolin and DNase I were then added to each sample.
  • Buffer A without additives, with 250 nM purified human gelsolin, 250 nM bovine pancreatic DNase I (Worthington Biochemical, Freehold, New Jersey) or with a 250nM combination of gelsolin and DNase I were then added to each sample.
  • DNase I caused a greater reduction in the viscosity of the sputum than did gelsolin alone. DNase I at the same concentration had no effect on the viscosity of the samples. The viscosity of sputum samples from the same patient that were treated with Buffer A alone did not vary over the course of the experiment.
  • Viscosity was measured in a torsion pendulum and values were computed from the rate of creep of the sample at a stress of 125 mPa, as described in P.A. Janmey, J. Biochem. Biophvs. Methods. 22:41 (1991). Uniform plugs of .25 ml were cut from coherent masses of the sputum samples and the plugs were layered onto round cover-slips. Ten microliters of Buffer A without additives or with a 250 nM combination o£ purified human gelsolin and Gc globulin (Calbiochem, La Jolla, California) were then added to each sample and the cover-slips were pressed against the suspended plate of the torsion pendulum. Duplicate measurements of the viscosity of the sputum sample from each patient were . made 15, 30, 45 and 60 minutes after the addition of the buffer.
  • gelsolin combined with Gc globulin rapidly reduced the viscosity of the sputum samples.
  • the viscosity of sputum samples from the same patient that were treated with Buffer A alone did not vary over the course of the experiment.

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Abstract

Procédés favorisant une circulation d'air normale dans les voies respiratoires de patients présentant une circulation d'air et un dégagement ciliaire entravés par la présence dans les voies respiratoires de substances pathologiques, notamment de mucosités. Des protéines se liant à l'actine sont administrées dans les voies respiratoires d'un patient présentant l'état respiratoire pathologique susmentionné. Ces protéines se lient aux polymères d'actine présents dans lesdites substances et en diminuent la viscosité. Ces protéines empêchent également que l'actine ne se lie à la DNase I exogène ou endogène, ce qui augmente la dégradation des polymères d'ADN dans les substances.
PCT/US1994/003667 1993-04-02 1994-04-04 Procede de reduction de la viscosite de mucosites pathologiques presentes dans les voies respiratoires WO1994022465A1 (fr)

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GB9520020A GB2293102B (en) 1993-04-02 1994-04-04 Use of actin-binding compounds for reducing the viscosity of pathalogical mucoid airway contents in the respiratory tract
CA002159205A CA2159205A1 (fr) 1993-04-02 1994-04-04 Methode pour abaisser la viscosite du contenu mucoide pathologique dans les voies respiratoires
AU66250/94A AU695033B2 (en) 1993-04-02 1994-04-04 Method for reducing the viscosity of pathological mucoid airway contents in the respiratoy tract
BR9405856A BR9405856A (pt) 1993-04-02 1994-04-04 Processo para redução de viscosidade de conteúdo de vias aéreas mocóides patológicos no trato respiratório
DE69419907T DE69419907T2 (de) 1993-04-02 1994-04-04 Verwendung von aktin-bindenden verbindungen zur herstellung eines medikaments zur verminderung der viskosität von pathalogischen schleimartigen bestandteilen der atemwege
JP6522448A JPH08510998A (ja) 1993-04-02 1994-04-04 呼吸道内の病理学的ムコイド気道内容物の粘度を低下させる方法
KR1019950704203A KR100308679B1 (ko) 1993-04-02 1994-04-04 호흡기내병리학적뮤코이드기도내용물의점도를감소시키는방법
DK94914023T DK0692970T3 (da) 1993-04-02 1994-04-04 Anvendelse af actin-bindende forbindelser til fremstilling af et lægemiddel til reduktion af viskositeten af patologisk sli
EP94914023A EP0692970B1 (fr) 1993-04-02 1994-04-04 Utilisation des composes se liant a l'actine pour la fabrication d'un medicament pour la reduction de la viscosite de contenus mucoides pathologiques presents dans les voies respiratoires
NO953862A NO953862D0 (no) 1993-04-02 1995-09-29 Fremgangsmåte for å redusere viskositeten i luftveienes patologiske mucusinnhold i den respiratoriske trakt
GR990402778T GR3031753T3 (en) 1993-04-02 1999-11-04 Method for reducing the viscosity of pathological mucoid airway contents in the respiratoy tract

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NO953862L (no) 1995-09-29
ATE182790T1 (de) 1999-08-15
CN1107521C (zh) 2003-05-07
DE69419907D1 (de) 1999-09-09
EP0692970B1 (fr) 1999-08-04
NO953862D0 (no) 1995-09-29
GB2293102A (en) 1996-03-20
BR9405856A (pt) 1996-03-05
DK0692970T3 (da) 1999-12-06
JP2001302537A (ja) 2001-10-31
ES2135576T3 (es) 1999-11-01
EP0692970A1 (fr) 1996-01-24
CA2159205A1 (fr) 1994-10-13
GB9520020D0 (en) 1995-12-06
US5464817A (en) 1995-11-07
AU695033B2 (en) 1998-08-06
SG49574A1 (en) 1998-06-15
JPH08510998A (ja) 1996-11-19
CN1122108A (zh) 1996-05-08
AU6625094A (en) 1994-10-24
GB2293102B (en) 1996-10-09
KR100308679B1 (ko) 2001-11-30
DE69419907T2 (de) 1999-12-09
US5656589A (en) 1997-08-12
GR3031753T3 (en) 2000-02-29

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